Bottom Line:
The neuron specific RNA-binding proteins NOVA1 and NOVA2 are highly homologous alternative splicing regulators.NOVA proteins regulate at least 700 alternative splicing events in vivo, yet relatively little is known about the biologic consequences of NOVA action and in particular about functional differences between NOVA1 and NOVA2.Thus we have discovered that NOVA2 uniquely regulates alternative splicing of a coordinate set of transcripts encoding key components in cortical, brainstem and spinal axon guidance/outgrowth pathways during neural differentiation, with severe functional consequences in vivo.

fig9s1: NOVA1 and NOVA2 expression in spiral ganglion (SG) and in superior olive neurons but not in hair cells.(A) Sagittal sections of Rosa26 (R26R); Nova2-/- mice at six month of age. β-gal staining shows that the expression of Nova2 was switched on during development in the spiral ganglion (SG) were the soma of the afferent innervating neurons are located (B) The enlarged organ of Corti area (dashed line in A) shows no expression of Nova2 in the cochlea, including IHC (red arrowhead) and OHC (green arrowhead). (C) Control Rosa26 (R26R); Nova2+/+ mouse shows no staining. (D) Immunostaining of P20 SG cells with an antiserum that recognizes NOVA1 and NOVA2 (E) showing the expression of NOVA1 in Nova2-/- mouse and demonstrating that NOVA1 and NOVA2 are expressed in SG. (F) In addition there is label in the soma of the medial olivococlear (MOC) neurons projecting efferent axons to the OHC which are located in the Medial superior olive (MSO) and lateral olivocochlear (LOC) neurons projecting axons the afferents below the IHC whose somas are located in the lateral superior olive (LSO) of the brainstem. (G) Enlarged area showing stained neuronal soma in MSO and LSO. (G–I) Immunostaining of a comparable section at P14 (H) wild-type section stained with C-16 a NOVA2 specific antibody. (I–J) Nova2-/- section stained simultaneously with C-16 antibody (showing unspecific background) and (I) with an antiserum that recognizes NOVA1 and NOVA2 showing the expression of NOVA1 in the superior olive. (K1–K3) Total RNA was extracted from micro dissected organ of Corti (dashed line rectangle), analysed by qPCR and normalized by actin. The plots show Nova1 and Nova2 gene expression relative to the expression of Nova1 in control P7 mouse at (K1) P7 and (K2) P14. (K3) the expression of the hair cell marker Myosin 7a (Myo7a) is shown at the indicated time points as a control. The reduced expression at P18 reflects problems in the dissection due to increased calcification. There is no apparent problem at P14, suggesting that the reduction in NOVA1 and NOVA2 expression at P14 is a biological phenomenon that probably reflects pruning of innervation. V: motor trigeminal nucleus, VII: Facial Nucleus. Scale bars; 200 μm (A), 50 μm (B) 10 μm (H–J). *p<0.05, **p<0.01 (n = 3, t test). Data are presented as mean ± SE.DOI:http://dx.doi.org/10.7554/eLife.14371.027

Mentions:
The inner ear is innervated by two types of fibers, afferent innervation consisting of both vestibular and cochlear (spiral ganglion neurons; Mao et al., 2014) and from efferent fibers derived from rhombomere 4 of the hindbrain (Simmons et al., 2011). Efferents are guided by and extend along afferents to reach the sensory epithelia of the ear arriving at approximately the same time (Fritzsch et al., 1998; Ma et al., 2000). Efferents are a unique population of ventral brainstem neurons derived from facial motoneurons that project to the inner ear in contrast to facial muscle fibers or glands (Karis et al. 2001). Some transcription factors have been hypothesized to be relevant for guiding inner ear efferents (Duncan and Fritzsch, 2013), however, essentially nothing is known about how these neurons diverge from facial motoneurons to selectively reach the ear to innervate hair cells and afferent processes (Simmons et al., 2011). Dye tracing analysis comparing with control mice at E14.5 (Figure 9A,A’) showed that Nova2 (Figure 9C,C’) but not Nova1 (Figure 9B,B’) was essential for proper progression of efferent growth along afferents. Moreover, although efferent neurons expressed both NOVA1 (Figure 9—figure supplement 1I–J) and NOVA2 (Figure 9—figure supplement 1F–H), Nova1 by itself was not capable of maintaining the function. In the absence of both Nova1/Nova2, the efferents stalled when they reached vestibular ganglion neurons (Figure 9D,D’) while afferent fibers that also expressed both NOVA1 and NOVA2 (Figure 9—figure supplement 1A–E, K) reached the cochlea normally. Our data suggested that Nova2 played a crucial role for efferent guidance relative to Nova1, but both cooperate for normal efferent fiber extension. Interestingly, vestibular efferent innervation which segregated from cochlear efferents at E14.5 (Bruce et al., 1997) was unaffected by the absence of Nova1 or Nova2 alone (see green efferents reaching the posterior canal crista (PC) in Figure 9B,C) but was also completely stalled in Nova1/Nova2 double knockouts (green label, Figure 9D). Postnatal Nova2-/- mice vestibular innervation was also normal (not shown).10.7554/eLife.14371.026Figure 9.NOVA2 expression is necessary for efferent innervation, targeting to the cochlea during embryonic development.

fig9s1: NOVA1 and NOVA2 expression in spiral ganglion (SG) and in superior olive neurons but not in hair cells.(A) Sagittal sections of Rosa26 (R26R); Nova2-/- mice at six month of age. β-gal staining shows that the expression of Nova2 was switched on during development in the spiral ganglion (SG) were the soma of the afferent innervating neurons are located (B) The enlarged organ of Corti area (dashed line in A) shows no expression of Nova2 in the cochlea, including IHC (red arrowhead) and OHC (green arrowhead). (C) Control Rosa26 (R26R); Nova2+/+ mouse shows no staining. (D) Immunostaining of P20 SG cells with an antiserum that recognizes NOVA1 and NOVA2 (E) showing the expression of NOVA1 in Nova2-/- mouse and demonstrating that NOVA1 and NOVA2 are expressed in SG. (F) In addition there is label in the soma of the medial olivococlear (MOC) neurons projecting efferent axons to the OHC which are located in the Medial superior olive (MSO) and lateral olivocochlear (LOC) neurons projecting axons the afferents below the IHC whose somas are located in the lateral superior olive (LSO) of the brainstem. (G) Enlarged area showing stained neuronal soma in MSO and LSO. (G–I) Immunostaining of a comparable section at P14 (H) wild-type section stained with C-16 a NOVA2 specific antibody. (I–J) Nova2-/- section stained simultaneously with C-16 antibody (showing unspecific background) and (I) with an antiserum that recognizes NOVA1 and NOVA2 showing the expression of NOVA1 in the superior olive. (K1–K3) Total RNA was extracted from micro dissected organ of Corti (dashed line rectangle), analysed by qPCR and normalized by actin. The plots show Nova1 and Nova2 gene expression relative to the expression of Nova1 in control P7 mouse at (K1) P7 and (K2) P14. (K3) the expression of the hair cell marker Myosin 7a (Myo7a) is shown at the indicated time points as a control. The reduced expression at P18 reflects problems in the dissection due to increased calcification. There is no apparent problem at P14, suggesting that the reduction in NOVA1 and NOVA2 expression at P14 is a biological phenomenon that probably reflects pruning of innervation. V: motor trigeminal nucleus, VII: Facial Nucleus. Scale bars; 200 μm (A), 50 μm (B) 10 μm (H–J). *p<0.05, **p<0.01 (n = 3, t test). Data are presented as mean ± SE.DOI:http://dx.doi.org/10.7554/eLife.14371.027

Mentions:
The inner ear is innervated by two types of fibers, afferent innervation consisting of both vestibular and cochlear (spiral ganglion neurons; Mao et al., 2014) and from efferent fibers derived from rhombomere 4 of the hindbrain (Simmons et al., 2011). Efferents are guided by and extend along afferents to reach the sensory epithelia of the ear arriving at approximately the same time (Fritzsch et al., 1998; Ma et al., 2000). Efferents are a unique population of ventral brainstem neurons derived from facial motoneurons that project to the inner ear in contrast to facial muscle fibers or glands (Karis et al. 2001). Some transcription factors have been hypothesized to be relevant for guiding inner ear efferents (Duncan and Fritzsch, 2013), however, essentially nothing is known about how these neurons diverge from facial motoneurons to selectively reach the ear to innervate hair cells and afferent processes (Simmons et al., 2011). Dye tracing analysis comparing with control mice at E14.5 (Figure 9A,A’) showed that Nova2 (Figure 9C,C’) but not Nova1 (Figure 9B,B’) was essential for proper progression of efferent growth along afferents. Moreover, although efferent neurons expressed both NOVA1 (Figure 9—figure supplement 1I–J) and NOVA2 (Figure 9—figure supplement 1F–H), Nova1 by itself was not capable of maintaining the function. In the absence of both Nova1/Nova2, the efferents stalled when they reached vestibular ganglion neurons (Figure 9D,D’) while afferent fibers that also expressed both NOVA1 and NOVA2 (Figure 9—figure supplement 1A–E, K) reached the cochlea normally. Our data suggested that Nova2 played a crucial role for efferent guidance relative to Nova1, but both cooperate for normal efferent fiber extension. Interestingly, vestibular efferent innervation which segregated from cochlear efferents at E14.5 (Bruce et al., 1997) was unaffected by the absence of Nova1 or Nova2 alone (see green efferents reaching the posterior canal crista (PC) in Figure 9B,C) but was also completely stalled in Nova1/Nova2 double knockouts (green label, Figure 9D). Postnatal Nova2-/- mice vestibular innervation was also normal (not shown).10.7554/eLife.14371.026Figure 9.NOVA2 expression is necessary for efferent innervation, targeting to the cochlea during embryonic development.

Bottom Line:
The neuron specific RNA-binding proteins NOVA1 and NOVA2 are highly homologous alternative splicing regulators.NOVA proteins regulate at least 700 alternative splicing events in vivo, yet relatively little is known about the biologic consequences of NOVA action and in particular about functional differences between NOVA1 and NOVA2.Thus we have discovered that NOVA2 uniquely regulates alternative splicing of a coordinate set of transcripts encoding key components in cortical, brainstem and spinal axon guidance/outgrowth pathways during neural differentiation, with severe functional consequences in vivo.